GSM6619760: GL2 (control) siRNA(replicate 2); Mus musculus; RNA-Seq
Total RNA extraction by using kit from Roche Library generation, quality control, and quantification:   500ng of total RNA per sample were utilized as input for mRNA enrichment procedure with 'NEBNext® Poly(A) mRNA Magnetic Isolation Module' (E7490L; New England Biolabs) followed by stranded cDNA library generation using 'NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina' (E7760L; New England Biolabs). All steps were performed as recommended in user manualE7760 (Version 1.0_02-2017; NEB) except that all reactions were downscaled to 2/3 of initial volumes.   cDNA libraries were barcoded by dual indexing approach, using 'NEBNext Multiplex Oligos for Illumina – 96 Unique Dual Index Primer Pairs' (6440S; New England Biolabs). All generated cDNA libraries were amplified with 7 cycles of final pcr.   One additional purification step was introduced at the end of the standard procedure, using 1.2x 'Agencourt® AMPure® XP Beads' (#A63881; Beckman Coulter, Inc.). Fragment length distribution of individual libraries was monitored using 'Bioanalyzer High Sensitivity DNA Assay' (5067-4626; Agilent Technologies). Quantification of libraries was performed by use of the 'Qubit® dsDNA HS Assay Kit' (Q32854; ThermoFisher Scientific).     Library denaturation and Sequencing run:   Equal molar amounts of individually barcoded libraries were pooled for a common sequencing run in which each analyzed library constituted 16.7% of overall flowcell / run capacity. The library pool was denatured with NaOH and was finally diluted to 1.8pM according to the Denature and Dilute Libraries Guide (Document # 15048776 v02; Illumina). 1.3ml of the denatured pool was loaded on an Illumina NextSeq 550 sequencer using a Mid Output Flowcell (130M cluster) for 2x75bp paired-end reads (20024904; Illumina). Sequencing was performed with the following settings: Sequence reads 1 and 2 with 76 bases each; Index reads 1 with 8 bases each; no index reads 2.